Probiotic composition based on the enterococcus strain and used as a treatment means and method for the production thereof



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Method for producing epithelial adhesive polypeptide In another embodiment there is provided a method for producing an epithelial adhesive polypeptide or a MALT cell adhesive polypeptide, or a fragment thereof, comprising the step of culturing a host cell as described herein under conditions suitable for the production of said epithelial adhesive polypeptide, or fragment thereof. The adhesive polypeptide is preferably selected from any of SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8, including functional equivalents and variants and fragments thereof.
Antibodies

There is also provided a polyclonal antibody or a monoclonal antibody specific for any of SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8, including functional equivalents and variants and fragments thereof.


Antagonists and agonists The invention also provided antagonists and agonists for any of SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8, including functional equivalents and variants and fragments thereof.
Pharmaceutical compositions and methods for treatment of an individual The is provided a pharmaceutical composition comprising a polypeptide selected from any of SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8, including functional equivalents and variants and fragments thereof. The polypeptide can be provided in purified or isolated form or the polypeptide can be provided as part of a Lactobacillus cell and/or Bifidobacterium cell in a composition comprising such cells.
Accordingly, the invention in preferred embodiments relates to pharmaceutical compositions which comprise the above-mentioned polypeptides as well as variants or fragments of these molecules as defined herein above for the treatment of disor- ders of the immune system.
Pharmaceutically and/or veterinary useful therapeutic compositions according to the invention can be formulated according to known methods such as by the admixture of one or more pharmaceutically or veterinary acceptable excipients or carriers.
Examples of such excipients, carriers and methods of formulation may be found e. g. in Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton, PA). To form a pharmaceutically or veterinary acceptable composition suitable for effective administration, such compositions will contain an effective amount of a polypeptide, nucleic acid, antibody or compound modulator.
Therapeutic or diagnostic compositions of the invention are administered to an

individual (mammal-human or animal) or used in amounts sufficient to treat or diagnose apoptosis-related disorders. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.


The term functional derivative includes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
Pharmaceutical and veterinary compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art. The therapeutically effective dose can be estimated initially either in cell culture assays, e. g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such infor- mation can then be used to determine useful doses and routes for administration in humans and other animals. A therapeutically effective dose refers to that amount of compound, peptide, antibody or nucleic acid which ameliorate or prevent a dysfunc- tional apoptotic condition. The exact dosage is chosen by the individual physician in view of the patient to be treated.
Compounds identified according to the methods disclosed herein as well as, therapeutic antibodies, therapeutic nucleic acids and peptides contemplated herein may be used alone at appropriate dosages defined by routine testing in order to obtain optimal modulation of living activity. In addition, co-administration or sequential administration of these and other agents may be desirable.
The pharmaceutical or veterinary compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular. Administration of pharmaceutical compositions is accomplished orally or parenterally.
Methods of parenteral delivery include topical, intra-arterial (directly to the tissue),

intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention.


The compositions containing compounds identified according to this invention as the active ingredient for use in the modulation of a protein which mediates apoptosis can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound, nucleic acid, or peptide desired can be employed as an apoptosis modulating agent.
The daily dosage of the products may be varied over a wide range such as e. g. from about 1 to 10,000 mg per adult human/per day. For oral administration, the compositions are preferably provided in the form of scored or unscored tablets containing 1.0, 25,50, 100,150, 250, 500,1000, 5000, and 10000 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day. The range is more par- ticularly from about 0.001 mg/kg to preferably less than 100 mg/kg of body weight per day.
Of course the dosage level will vary depending upon the potency of the particular compound. Certain compounds will be more potent than others. In addition, the dosage level will vary depending upon the bioavailability of the compound. The more bioavailable and potent the compound, the less compound will need to be administered through any delivery route, including but not limited to oral delivery.
The dosages of living modulators are adjusted when combined to achieve desired effects. On the other hand, dosages of these various agents may be independently

optimised and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibi- tors.


There is also provided combination therapies comprising the step of administering the vaccine compositions according to the invention in combination with a chemo- therapeutic agent and/or an immunotherapeutic agent and/or a cancer vaccine.
There is also provided a method of treating an individual with the above methods in order to alleliorate, cure or prophylactically treat an individual suffering from a condi- tion affecting the immune system of the individual.
Conditions capable of being treated include, but is not limited to auto-immune dis- eases. Autoimmune diseases may be loosely grouped into those primarily restricted to specific organs or tissues and those that affect the entire body. Examples of organ-specific disorders (with the organ affected) include multiple sclerosis (myelin coating on nerve processes), type I diabetes mellitus (pancreas), Hashimotos thy- roiditis (thyroid gland), pernicious anemia (stomach), Addison's disease (adrenal glands), myasthenia gravis (acetylcholine receptors at neuromuscular junction), rheumatoid arthritis (joint lining), uveitis (eye), psoriasis (skin), Guillain-Barre Syn- drome (nerve cells) ana'Grave's disease (thyroid). Systemic autoimmune diseases include systemic lupus erythematosus and dermatomyositis.
Other examples of hypersensitivity disorders capable of being treated in accordance with the present invention include asthma, eczema, topical dermatitis, contact dermatitis, other eczematous dermatitides, seborrheic dermatitis, rhinitis, Lichen planus, Pemplugus, bullous Pemphigoid, Epidermolysis bullosa, uritcaris, an- gioedemas, vasculitides, erythemas, cutaneous eosinophilias, Alopecia areata, atherosclerosis, primary biliary cirrhosis and nephrotic syndrome. Related diseases include intestinal inflammations, such as Coeliac disease, proctitis, eosinophilia gastroenteritis, mastocytosis, inflammatory bowel disease, Crohn's disease and ulcerative colitis, as well as food-related allergies.

Methods for performing quality control and strain development In yet further embodiments there is provided a method for determining the probiotic potential of a microorganism, said method comprising the steps of i) determining the relative production and/or amount in the microorganism of a microbial cell surface polypeptide the intracellular equivalent of which is selected from glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, triose phosphate isomerase, and enolase, including variants and functional equivalents thereof, and ii) comparing the relative production of the microbial cell surface polypeptide to the production in L. plantarum 299v of a cell surface polypeptide having the same activity under substantially identical growth conditions.


There is also provided a method for optimising the probiotic potential of a microbial cell, said method comprising the steps of i) obtaining a microbial cell the probiotic potential of which is to be optimised, and iii) optimising the production and/or secretion and/or modification in the microbial cell of a polypeptide selected from the group consisting of glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, triose phosphate isomerase, and enolase, including variants and functional equivalents thereof, and thereby optimising the probiotic potential of the cell.
Examples Example 1 : Isolation of major surface located proteins from Lactobacillus plantarum strain 299v Lactobacillus plantarum strain 299v was pre-cultivated on Man-Rogosa-Sharpe (MRS) (Oxoid, Basingstoke, Hampshire, England) agar plates for 48 hours at 37 C.

Glass tubes containing 15 mL MRS medium was subsequently inoculated with pre- cultured cells of L. plantarum 299v and left overnight at 37 C without aeration.


The L. plantarum 299v culture (OD600 of 6.5) was harvested by centrifugation (4000 x g/4 C) and washed once in PBS buffer (136.9 mM sodium chloride, 2.68 mM potassium chloride, 8.1 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate). The pellet was resuspended in PBS to a final optical density at 600 nm of 65. The suspension was incubated statically at 37 C for 3 hours, and proteins released from the outer cell surface of L. plantarum 299v into the PBS buffer was analysed by SDS-PAGE.
Samples were analysed by SDS-PAGE using 12 % Invitrogen NuPage BIS-TRIS gels (Invitrogen, San Diego, Calif.), gelmatrix : acrylamide/bis-acrylamide, size: 8x8 cm2, 1 mm gel thickness. The running buffer used was a 2- (N- morpholino) ethanesulfonic acid (MES) SDS buffer. Samples for SDS-PAGE analysis were prepared by mixing 22.5 l sample, 12. 5 lli NuPage lithium dodecyl sulphate (LDS) sample buffer (Invitrogen) and 5 I NuPageTM sample reducing agent (invitro- gen). The mixture was incubated at 56 C for 20 min prior to analysis. 20 uL of the sample was analysed in parallel with 10 pL of a Precision Plus ProteinTM Stan- dards, All Blue molecular weight standard (Bio-Rad Laboratories, CA, USA). Gels were run for 40 min at 200 V. Protein-bands were visualised by Coomassie blue staining (Bio-SafeTM Coomassie, Bio-Rad Laboratories). SDS-PAGE analysis of outer surface associated proteins revealed one distinct band of approximately MW 38.5 kDa and two faint bands of approximately MW 51 kDA and 100 kDA (Fig. 1).
Example 2: Analysis of released surface proteins by mass spectrometry Bands visualised by Coomassie blue staining were excised from the gel and in-gel digested with trypsin. The excised gel bands were transferred to 1.5 mL eppendorf microcentrifuge tubes and incubated with 200 lui ultra pure water. After 10 min of incubation the gel pieces were transferred to a clean glass plate, cut into small pieces (approximately 1 mm3) and rinsed with 200 pu ultra pure water in eppendorf microcentrifuge tubes. The gel pieces were rinsed, shrunk by adding 30 lli 100% acetonitrile, and subsequently dried in a vacuum centrifuge. Then the gel pieces were swollen in a digestion buffer, 50 mM NH4C03, 12.5 ng/lli trypsin (Promega,

Madison, Wl, USA, modified, sequencing grade) in an ice-cold water-bath. After 45 min the supernatant was removed and replaced with 30 uL 50 mM (NH4) 2CO3 buffer. Enzymatic cleavage was performed overnight at 37 C. The protein digests were loaded on POROS R2 (Applied Biosystems, Calif., USA) reverse phase column material prepared in GELoader tips (Eppendorf, Hamburg, Germany) prepared as described (Kussmann et al. ; 1997). The column was washed with 50 uL formic acid and the bound peptides were eluted directly into a nanospray needle (Protana, Odense, Denmark) with 2uLofa 50% MeOH-1 % formic acid solution. The digestion mixtures were analysed by nanoelectrospray mass spectrometry (nano ESI MS) using a Q-Tof mass spectrometer (Micromass, Manchester, United Kingdom). Selected peptides were sequenced by nanoelectrospray tandem mass spectrometry (nano ESI MS/MS). The resulting peptide sequences were used to search for short nearly exact matches in the non-redundant Blast protein-protein (www. ncbi. nim. nih/gov/BLAST/) sequence database, enabling identification of the proteins.


Example 3: Identification of surface proteins from L. plantarum SDS-PAGE analysis of outer surface associated proteins revealed one distinct band of approximately MW 38. 5 kDa and two faint bands of approximately MW 51 kDA and 100 kDA (Fig. 1). The three protein bands were excised and in-gel digested with trypsin. The resulting tryptic digests were analysed using nano ESI MS and peptides of interest were selected and sequenced by nano ESI MS/MS analysis. This is shown in Fig. 2 with the tryptic digest of the band at MW 38. 5 kDa as an example.
Fig. 2a shows the ESI MS analysis of the tryptic digest. The double charged peptide at m 918. 96 Da (Fig. 2b) was sequenced by ESI MS/MS analysis (Fig. 2c). The peptides selected for nano ESI MS/MS analysis, the resulting sequences and protein protein identities from the MS analysis of the tryptic digest of the protein bands at MW 38.5 and 51.0 kDa are summarised in Table 1.
Table 1. Peptide sequences and protein identifications/assignments from proteins derived from one-dimensional SDS-PAGE separation of cell surface associated proteins. The bands were in-gel digested with trypsin and selected peptides sequenced by ESI MS/MS analysis. Proteins were identified using the non-redundant Blast protein-protein (www. ncbi. nim. nih/aov/BLAST/) sequence database.
MW of gel band ions measured Peptide (s) Identification 38. 96 (2+) VYNVNDDLLTADDR Glyceraldehyde (SEQ ID NO : 12) Dehydrogenase 38. (2+) VDGSLTE (I/L) VA (I/L) DK Glyceraldehyde (SEQ ID : 13) Dehydrogenase 38. (2+) V (I/L) (I/L) (SEQ ID : 14) Dehydrogenase 51. 6 (1+) VGDD (I/L)

Enotase (SEQ ID NO : 15) 51. 8 (2+) VGYDVTDQR (SEQ ID NO : 16) 51. 61 (1+) DTTLAD (I/L) (SEQ ID : 17) 51. 4 (2+) STAVGDEGGFA ase (SEQ ID NO : 18) 51. 0 5 (2+) PTVEVE (I/L) (SEQ ID NO : 19) 51. 0 9 (2+) NALLGVS Enolase (SEQ ID : 20) 100. 0 05 (2+) TAQWGTYNAGET-Murimidase

VYYNKD (SEQ ID NO : 29) sequenceExample 4: Cloning of the gene encoding glyceraldehyde 3 phosphate dehydrogenase (GAPDH) from Lactobacillus plantarum 299v The result of the blastp analysis described in example 3 clearly identified the ana- lyzed surface protein as GAPDH from L. plantaruni 299v.
Two degenerate primers GPD-Nterm (INGFGRIG (SEQ ID NO : 21) ) (5'ATHAAYGGNTTYGGNMGNATHGGN 3' (SEQ ID NO : 22) ) and GPD-mid REV (TGAAKAVGK (SEQ ID NO : 23)) (5'YTTNCCNACNGCYTTNGC NGCNCCNGT 3' (SEQ ID NO : 24)) which have been used for PCR amplification of the gapdh gene from Mucor circinelloides (Wolff & Arnau; 2002) were used to amplify an internal 0.7 kb fragment of the gapdh gene from L. plantarum 299v. The following PCR profile was used to amplify the 0.7 kb gapdh fragment:

94 C 2 min 94 55 72 C 30 sec 72 C 7 min C 30 secTotal DNA from L. plantarum 299v was used as template. A standard PCR reaction condition with the Taq DNA polymerase (Invitrogen, Carlsbad, Calif.), expect that the concentration of each primer was 5 uM, was used to amplify the gapdh gene.


A PCR product of the expected size was purified from an agarose gel using the GFXTM PCR DNA and gel band purification kit (Amersham Biosciences Corp. , Pis- cataway, NJ) and inserted into the pCR;2. 1-TOPO vector (Invitrogen, Carlsbad, Calif.). The DNA sequence of the insert was determined using an ALFexpress DNA sequence and universal M13 forward and reverse primers. The remaining part of the gapdh gene and the adjacent DNA regions were amplified by consecutive rounds of inverse PCR (Ochman et al. ; 1988). In short, total DNA of L. plan arum 299v was digested with either EcoRl or Hindlil and related in a large volume. PCR amplifications were carried out using DNA primers based on DNA sequences that were obtained during the successive rounds of inverse PCR. The polynucleotide sequence and the polypeptide sequence of gapdh are shown in Figs. 3 and 4. The gapdh gene of 299v encodes a 340 aa protein. A blastp similarity search showed that gapdh protein of L. plantarum is 96% (low complexity filter on) identical to the gapdh gene from L. plantarum WCFS1 (Acc. No. CAD63377) and 81% (low com- plexity filter on) identical to a hypothetical protein from Lb. gasseri (Acc. No.
ZP00047412. 1).
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2. 1.12) is an enzyme of the glycolytic pathway, in which it catalyzes the oxidative conversion of D- glyceraldehyde 3-phosphate and phosphate to 3-phospho-D-glyceroyl phosphate using NAD+ as an acceptor.
Example 5: Cloning of the gene encoding phosphoglycerate kinase (PGK) from Lactobacillus plantarum 299v

During the course of cloning and sequencing of the gapdh gene we identified the 5' end of the gene encoding phosphoglycerate kinase (PGK; EC. 2.7. 2. 3.). In the glycolytic pathway, PGK catalyses the phosphotransferase reaction between 3phospho-D-glyceroyl phosphate and ADP to produce ATP and 3-phospho-Dglycerate. The pgk gene is located immediately downstream of the gapdh gene of L. plantarum 299v. The remaining part of the pgk gene was cloned by consecutive rounds of inverse PCR as described above. The polynucleotide sequence and the polypeptide sequence of pgk are shown in Figs. 3 and 5. The pgk gene encodes a 400 aa protein. A blastp similarity search showed that the pgk protein from L. plantarum is 99% (low complexity filter on) identical to the pgk gene from L. plantarum WCFS1 (Ace. No. CAD63378) and 74% (low complexity filter on) identical to the pgk gene from Lb. delbrueckii (Acc. No. CAD56495).


Example 6: Cloning of the gene encoding triosephosphate isomerase (TPI) from Lactobacillus plantarum 299v During the course of cloning and sequencing of the pgk gene we identified the 5' end of the gene encoding triosephosphate isomerase (TPI ; EC. 5.3. 1. 1. ). The tpi gene is located immediately downstream of the pgk gene in L. plantarum 299v. The remaining part of the tpi gene was cloned by consecutive rounds of inverse PCR as described above. The polynucleotide sequence and the polypeptide sequence of tpi are shown in Figs. 3 and 6. Tpi encodes a 252 aa protein. A blastp similarity search showed that the tpi protein from L. plantarur1 is 99% (low complexity filter on) identical to the tpi gene from Lactobacillus p/anfamm WCFS1 (Ace No. CAD63379) and 71% (low complexity filter on) identical to the tpi gene from L. delbruecleii (Acc. No.
032757).
TPI catalyzes the isomerisation of D-glyceraldehyde 3-phosphate to glycerone phosphate and vice versa in the glycolytic pathway.
Example 7: Cloning of the gene encoding enolase (eno) (phosphoenolpyruvate hydratase) from Lactobacillus plantarum 299v The result of the blastp analysis described in example 3 obviously identified the analysed surface protein as enolase from L. plantarum 299v. Enolase (2-phosphoD-glycerate hydrolyase ; EC 4.2. 1.11) catalyses the dehydration of 2phosphoglycerate to phosphoenolpyruvate.

Based on the amino acid sequences described in example 3 we designed two de- generate primers Eno-Deg1 (5'GTNGARGTNGARYTNTAYACNGA 3' (SEQ ID NO : 25) ) and Eno-Deg2 (5'RTTNGTNACRAANARRTCRTCNCC 3' (SEQ ID NO : 26)). Eno-Deg1 corresponds to the peptide sequence VEVELYTES (SEQ ID NO : 27), which, is found in the amino terminal of the enolase whereas Eno-Deg2 corresponds to the peptide sequence GDDLFVTN (SEQ ID NO : 28) located ap- proximately 300 amino acids downstream of the start codon of the enolase. The following PCR profile was used to amplify an internal 0.9 kb enolase fragment:


94 C 2 min 94 58 30 sec 10 72 C 45 sec 94 54 C 30 sec 10 72 C 45 sec 94 50 C 30 sec 10 72 C 45 sec 94 46 C 30 sec 15 72 C45sec" 72 C 30 secTotal DNA from L. planfarum 299v was used as template. A standard PCR reaction condition with the Taq DNA polymerase, expect that the concentration of each primer was 5 ; j. M, was used to amplify the internal fragment of the enolase gene.
A PCR product of approximately 0.9 kb was purified from an agarose gel using the GFXTM PCR DNA and gel band purification kit (Amersham Biosciences Corp. , Pis- cataway, NJ) and inserted into the pCR;2. 1-TOPO vector. The DNA sequence of the insert was determined using the ALFexpress DNA sequencer and universal M13 forward and reverse primers. The remaining part of the enolase gene and the adja- cent DNA regions were amplified by consecutive rounds of inverse PCR as de- scribed above. Using this strategy we identified the 3'end of triosephosphate iso- merase gene upstream of the 5'end of the enolase gene, which consequently re-

vealed that the four genes are placed in the order gapdh-pgk-tpi-eno and suggests that the genes are clustered in an operon.


The polynucleotide sequence and the polypeptide sequence of enolase are shown in Figs. 3 and 7. Enolase of 299v encodes a 442 aa protein. A blastp similarity search showed that enolase protein of L. plantarum 299v is 98% (low complexity filter on) identical to the phosphopyruvate hydratase from L. plantarum WCFS1 (Acc No. CAD63380) and 77% identical to a hypothetical protein from Lb. gasseri (Acc.
No. ZP00047409).
Example 8: Cloning of the gene encoding a putative regulator of the gapdh- pgk-tpi-eno operon from Lactobacillus plantarum 299v During the course of cloning and sequencing of the gapdh gene we identified the 3' end of the gene encoding a putative glycolytic regulator. The regulator gene is located upstream of the gapdh gene of L. plantarum 299v. The remaining part of the regulator gene was cloned by consecutive rounds of inverse PCR as described above. The polynucleotide sequence and the polypeptide sequence of the regulator are shown in Figs. 3 and 8. The glycolytic regulator encodes a 343 aa protein. A blastp similarity search showed that the glycolytic regulator from L. planŠ½arum 299v is 93% (low complexity filter on) identical to the central glycolytic regulator from L. plantarum WCFS1 (Acc. No. CAD63376) and 45% (low complexity filter on) identical to a hypothetical transcriptional regulator from Listeria innocua cc. No.
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